Breast cancer susceptibility gene GT198 and uses thereof

ABSTRACT

It has been discovered that the human GT198 gene (gene symbol PSMC3IP) at chromosome 17q21 acts as a tumor suppressor. The mutation of the GT198 gene causes the increased dominant negative splice variant activity and leads to the loss of wild type GT198 function, and in turn, induces breast and ovarian cancers. One embodiment provides compositions and methods for treating or alleviating one or more symptoms associated with cancer due to the GT198 gene mutations. Another embodiment provides methods and compositions for detecting cancer due to the mutation of the GT198 gene. Still another embodiment provides methods for identifying compounds, antibodies and natural product molecules that are useful for treating cancer due to the mutations of the GT198 gene. Preferably the disclosed compositions antagonize or interfere with the biological activity of splice variants of GT198.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of pending U.S. application Ser. No.12/763,483, filed Apr. 20, 2010, which claims priority to and benefit ofU.S. Provisional Patent Application No. 61/212,974 filed on Apr. 20,2009, and is incorporated herein by reference in its entirety.

REFERENCE TO SEQUENCE LISTING

The Sequence Listing submitted Jun. 19, 2013 as a text file named“KOLAN_101_CON.ST25.txt,” created on Jun. 19, 2013, and having a size of13,420 bytes is hereby incorporated by reference pursuant to 37 C.F.R.§1.52(e)(5).

FIELD OF THE INVENTION

Aspects of the invention are generally related to the field of molecularbiology, gene diagnostics, and gene therapy.

BACKGROUND OF THE INVENTION

Cancer is an often fatal disease that affects a significant portion ofthe population. The National Cancer Institute estimated that theage-adjusted death rate due to cancer in the U.S. was 192.7 per 100,000men and women per year. In January of 2003 approximately 10.5 millionAmericans had a history of cancer. Breast cancer is the most commonmalignancy in women, and is a major cause of mortality in women over 45years of age, especially in United States. Each year over 185,000 newcases are diagnosed and more than 40,000 women die of the disease.However, only a very small percentage of breast and ovarian cancers isattributable to the inheritance of mutations in cancer susceptibilitygenes such as BRCA1 and BRCA2. The majority of breast and ovariancancers require the knowledge of additional breast cancer genes for thediagnosis and treatment.

Cancer is a group of diseases characterized by uncontrolled growth andspread of abnormal cells. If the spread is not controlled, it can resultin death. Cancer is caused by both external factors (tobacco, chemicals,radiation, and infectious organisms) and internal factors (inheritedmutations, hormones, immune conditions, and mutations that occur frommetabolism). The regulation of gene expression involved in cancerdevelopment has been heavily investigated, but therapeutics and methodsfor detecting cancer are still needed.

Germline mutations in the BRCA1 and BRCA2 genes account for increasedsusceptibility to familial breast and ovarian cancers (Nathanson, K. L.,et al., Nat Med, 7:552-6 (2001)). BRCA1 encodes an 1863-amino acidprotein with an N-terminal RING domain facilitating ubiquitination and aC-terminal BRCT domain stimulating transcriptional activation (Welcsh,P. L., et al., Trends Genet, 16:69-74 (2000)). The BRCT domain inducesthe cleavage of RNA polymerase II upon ionizing radiation (Bennett, C.B. et al., PLoS ONE, 3:e1448 (2008)). The sequence encoded by the largeexon 11 of BRCA1 binds to Rad51, a protein critical for homologousrecombination and DNA-damage response (Chen, J. J., et al., Cancer Res,59:1752s-1756s (1999)). Alternative splicing variants of BRCA1, such asBRCA1Δ11b and BRCA1-IRIS (Wilson, C. A. et al., Oncogene, 14:1-16(1997); ElShamy, W. M. & Livingston, D. M., Nat Cell Biol, 6:954-67(2004)), have been identified with potential functional impact on BRCA1.

BRCA2 encodes a 3418-amino acid protein and has a very similar tissueexpression pattern to BRCA1 (Chodosh, L. A., J Mammary Gland BiolNeoplasia, 3:389-402 (1998)). Its large exon 11 encodes eight sequencerepeats called the BRC repeats, six of which interact with Rad51 (Bork,P., Blomberg, N. & Nilges, M., Nat Genet, 13:22-3 (1996); Bignell, G.,et al., Hum Mol Genet, 6:53-8 (1997); Davies, A. A. et al., Mol Cell,7:273-82 (2001)). Crystal structure analysis demonstrated that the BRCrepeat mimics a motif between the interfaces of Rad51 oligomerization(Pellegrini, L. et al., Nature, 420:287-93 (2002)), and that the bindingof BRCA2 to Rad51 is essential for both functions (Gudmundsdottir, K. &Ashworth, A., Oncogene, 25:5864-74 (2006)). The BRCA2 transcripts alsoundergo complex alternative splicing, and its splicing products are farfrom defined due to its large gene size (Speevak, M. D., et al., Eur JHum Genet, 11:951-4 (2003); Bieche, I. & Lidereau, R., Cancer Res,59:2546-50 (1999)).

The close functional relationship between BRCA1 and BRCA2 suggests theinvolvement in DNA-repair pathways in breast and ovarian cancers.However, the specific risk to breast and ovarian cancers that areevidently linked to hormone regulation has not been adequatelyexplained. In addition, early linkage analysis at the chromosome 17q21locus has provided substantial evidence for breast and ovarian cancerpredisposition that is inherited in a Mendelian fashion in families withearly onset cancers (Hall, J. M. et al., Science, 250:1684-9 (1990);Hall, J. M., et al., Am J Hum Genet, 50:1235-42 (1992); Narod, S. A., etal., Lancet, 338:82-3 (1991)). However, the BRCA1 mutations explainedonly a proportion of families with 17q21 association (Nathanson, K. L.,et al., Nat Med, 7:552-6 (2001); Miki, Y., et al., Science, 266, 66-71(1994)). This paradoxical phenomenon led to the speculation of thepresence of an additional candidate gene within the BRCA1 locus(Vogelstein, B. & Kinzler, K. W., Cell, 79:1-3 (1994)). In 1995, duringrefined locus mapping near BRCA1 at 17q21, GT198 (genomic transcript198, gene symbol PSMC3IP, also known as TBPIP or Hop2) was identified asa cDNA clone (Rommens, J. M., et al., Genomics, 28:530-42 (1995)). GT198was later characterized as a nuclear receptor coregulator that interactswith nuclear receptors and is involved in estrogen, androgen andprogesterone receptor-mediated gene regulation (Ko, L., et al., Mol CellBiol, 22, 357-69 (2002); Satoh, T., et al., Endocrinology, 150:3283-90(2009)). GT198 was also found to be homologous to yeast Hop2 (Petukhova,G. V., et al., Dev Cell, 5:927-36 (2003)), to interact with Rad51 and tostimulate DNA strand exchange in homologous recombination (Enomoto, R.,et al., J Biol Chem, 281:5575-81 (2006); Pezza, R. J., et al., GenesDev, 21:1758-66 (2007); Enomoto, R., et al., J Biol Chem, 279:35263-72(2004)).

Existing BRCA1 and BRCA2 genes for detecting breast and ovarian cancerand treating cancer are typically insufficient, especially for a largeamount of sporadic cancers.

Thus, it is an object of the invention to provide methods andcompositions for the early detection or diagnosis of cancer, for examplebreast and ovarian cancer.

It is another object of the invention to provide compositions andmethods for the treatment of one or more symptoms associated withcancer.

It is still another object to provide methods for screening for chemicalcompounds and small biological molecules such as antibodies that inhibitor alleviate pathologies due to cells having one or mutations resultingin cancer.

It is another embodiment to provide biomarkers for the detection anddiagnosis of cancer.

SUMMARY OF THE INVENTION

GT198 and alternative splice variants thereof are useful biomarkers forthe detection and diagnosis of cancer, preferably ovarian and breastcancer. It has been found that the GT198 gene at 17q21 has strikingsimilarities to BRCA1 in its regulation and in function. One embodimentprovides a method for detecting or assisting in the diagnosis of cancerby determining the presence of one or more mutations, alterations, orrearrangements of the GT198 gene in a biological sample obtained from asubject. In certain embodiments, the determining step is done bycontacting the sample with a probe specific for the one or moremutations, alterations, or rearrangements of the GT198 gene or GT198gene product to form a detectable complex between the probe and theGT198 gene or gene product. The presence of the detectable complex inthe biological sample is indicative of cancer. Preferred mutations inGT198 include, but are not limited to a substitution in exon 4 of GT198,a mutation at nucleotide 85 of exon 4, a mutation at nucleotide 88 ofintrons 4 of GT198, or a mutation at nucleotide 31 of the 5′untranslated region of GT198. Preferred GT198 variants include but arenot limited to GT198a, GT198-1, GT198-2, GT198-3, GT198-4, and GT198a-4.

Another embodiment provides methods and compositions for detectingcancer due to gene mutations and copy number losses in GT198 or cellscontaining increased splice variants of GT198 protein. The GT198mutations can be detected using PCR, conformation-sensitive gelelectrophoresis and direct sequencing approaches. The copy numberchanges can be detected by quantitative real-time PCR, southernblotting, and genomic walking, fluorescent in situ hybridizationmethods. An exemplary method for detecting GT198 variant proteinexpression in cancer tissues includes, but is not limited toimmunohistochemistry analysis. The detection of one or more GT198mutation that increases the variant expression of GT198 is indicative ofcancer.

Another method for diagnosing or assisting in the diagnosis of cancerincludes determining cytoplasmic expression levels of GT198 in a sampleobtained from a subject by contacting the sample with a probe specificfor GT198 to form a detectable complex between the probe and the GT198gene or gene product, wherein elevated cytoplasmic expression levels ofGT198 relative to a control is indicative of cancer.

Still another embodiment provides methods for treating cancer usingbiological molecules that inhibit, reduce, block or prevent GT198 andGT198 variant protein from functioning. Alternatively, these compoundscan reduce or inhibit the bioavailability of GT198 or variants thereof.Preferably the disclosed compositions antagonize or interfere withbiological activity of GT198 and/or its alternative splicing variantsincluding GT198a in cells with ectopic expression of GT198 variantproteins.

Another embodiment provides a method for screening of synthetic ornatural compounds that inhibit the activity of GT198 or an alternativelyspliced variant thereof. The method includes screening of antibodies orsmall nucleic acid molecules as such siRNA, microRNA, aptamer, andpeptide nucleic acid to inhibit the activity of GT198 or alternativelyspliced variants.

Kits are also provided. An exemplary kit includes a container containinga nucleic acid probe having at least 10 nucleotides that hybridize understringent conditions to a nucleic acid encoding a GT198 variant proteinand does not hybridize under stringent conditions to a nucleic acidencoding wild-type GT198 protein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1a is schematic representation of BRCA1, variant BRCA1Δ11b, BRCA2,GT198, and variant GT198a structures in which the BRC repeat-containingsequences are shown as black boxes. GT198 N-terminal and leucine zipperdomains are shown as open boxes. FIG. 1b is a sequence alignment of BRC3(SEQ ID NO:1) and BRC4 (SEQ ID NO:2) in BRCA2 and GT198 C-terminaldomain (SEQ ID NO:3) using ClustalW 2.0.8. Residues with homology to theBRC repeat of BRCA2 are boxed. Identical residues are indicated with anarrow, and the remaining shaded residues are conserved. Number indicatesamino acids. FIG. 1c is a diagram showing that the splice variants ofGT198 or BRCA1 or BRCA2 serve dominant negative roles to their wildtypes. GT198 potentially competes with BRCA2 in Rad51 (circle)interaction.

FIG. 2a is a schematic representation of the human GT198 variantsidentified by rapid amplification of 5′ complementary DNA ends (5′ RACE)and sequencing (not to scale). Mammalian GT198 has 8 exons. Introns areshown as lines, exons as bars, protein coding regions as thick bars.Deduced translation start and stop codons are indicated. Primerpositions are shown at the top. FIGS. 2b-e are a bar graphs of relativemRNA levels in undifferentiated P19 stem cells (EC) induced with 500 nMretinoic acid (RA) up to 4 days to form embryoid bodies (EB2-EB4).Relative mRNA levels of BRCA1 (FIG. 2b ), BRCA1Δ11b (FIG. 2c ), GT198(FIG. 2d ), or GT198a (FIG. 2e ) were obtained using real-timepolymerase chain reaction. Results are shown as means±s.e.m. of relativemRNA level.

FIG. 3a is a bar graph of transcriptional activity in P19 cellstransiently transfected with the indicated together with anMMTV-luciferase reporter (100 ng) and a glucocorticoid receptor (10 ng)that binds to the MMTV promoter. Cells were induced in the presence(solid bars) or the absence (clear bars) of ligand dexamethasone (100nM) overnight and the luciferase activity was measured by a Dynexluminometer. FIG. 3b is a bar graph of transcriptional activity of P19cells transfected as in FIG. 3a except using an increasing amount ofplasmids—0.0 ng (clear bars), 50 ng (shaded bars), 100 ng (hatchedbars), and 200 ng (solid bars) and induced by dexamethasone (100 nM).Luciferase activities shown are means of triplicate transfections±s.e.m.(n=3).

FIG. 4a is a schematic diagram of the GT198 gene. Introns are shown aslines and exons as boxes, Alu and L2 repeats as open orientatedtriangles, translation start and stop codons are indicated. Filledarrows indicate mutations and open arrows indicate locations of singlenucleotide polymorphisms (SNPs). FIG. 4b is a table summarizing the yearof onset, allelic mutations and SNPs (rs2292751, rs2292752) identified.FIG. 4c is a pedigree of the families of case 1, case 3, and case 9 withbreast cancer (filled circles and squares), other types of cancer(shaded circles and squares), or healthy (clear circles and squares).Circles represent women and squares represent men. Numbers insideindicate additional healthy siblings. Slash symbols indicate deceasedindividuals. Cancer types, year of onset, and year of death (d) areshown as available (BC, breast cancer; CRC, colorectal cancer; ST,stomach cancer; SK, skin cancer; LI, liver cancer; ES, esophagus cancer;PC, prostate cancer; LC, lung cancer).

DETAILED DESCRIPTION OF THE INVENTION I. Diagnostics for and Methods ofDiagnosing Cancer

GT198 and alternative splice variants thereof are useful biomarkers forthe detection and diagnosis of cancer, preferably ovarian and breastcancer. Antagonists of biological activity or bioavailability of GT198and variants thereof can be used to treat one or more symptoms ofcancer. In one embodiment, cytosolic expression of GT198 is indicativeof cancer. In another embodiment, expression of a GT198 splice variantis indicative of cancer. Preferred GT198 variants include, but are notlimited to GT198a, GT198-1, GT198-2, GT198-3, GT198-4, and GT198a-4.Compositions for treating cancer can be identified by screening forcompounds that inhibit the biological activity of GT198 or variantsthereof.

A. GT198

GT198 was originally identified as a transcript when the BRCA1 locus wassearched for additional breast cancer genes (Rommens, J. M., et al.,Genomics, 28:530-42 (1995)). The GT198 gene is located 470 Kb proximaland downstream to BRCA1, and 15 Kb distal to HSD17B1, which is one ofthe most closely linked markers identified during early linkage analysisbefore positional cloning of BRCA1 (Anderson, L. A., et al., Genomics,17:618-23 (1993); Black, D. M., et al., Am J Hum Genet, 52:702-10(1993)). The human GT198 gene is located between HSD17B1 and BRCA1 genesat chromosome 17q21. It is 15 Kb distal to HSD17B1 and 470 Kb proximalto BRCA1. Mammalian GT198 spans 5 Kb and encodes a 217-amino acidprotein containing an N-terminal domain and a C-terminal DNA-bindingdomain (DBD) (Enomoto, R., et al., J Biol Chem, 279:35263-72 (2004)),linked by a leucine zipper domain required for GT198 dimerization andprotein interaction (Ko, L., et al., Mol Cell Biol, 22, 357-69 (2002)).

The primary sequence of GT198 shares homology with the BRC repeats inBRCA2 which provides strong evidence for its interaction with Rad51.Endogenous expression of GT198 protein closely parallels that of BRCA1and BRCA2, which suggests they may function in the same pathways. GT198has dual transcriptional start sites, a feature also presents in BRCA1,permitting differential expression of the wild type and its splicevariant transcripts. GT198 variants encode a truncated proteincontaining the BRC repeat homology and inhibit wild type GT198 activity.Normal stem cell differentiation is accompanied with decreased variantand increased wild type expression in both GT198 and BRCA1. Ectopicexpression of GT198 variants, however, induces apoptosis, blocks Rad51foci formation and is found in cytoplasm of cells in primary breast andovarian cancer tissues when hundreds of cases were analyzed byimmunohistochemistry. Consistent with the above, variant BRCA1Δ11b ofBRCA1 was known to express in cytoplasm in breast cancers (Wilson, C.A., et al., Oncogene, 14, 1-16 (1997)). Furthermore, it is now beenfound that germline mutations of GT198 in early onset familial breastcancer patients. One of them with onset at age of 33 years has anonsense mutation generating a premature stop codon that will preventthe expression of full length GT198 but permit the expression of GT198variant. The same mutation was also carried by her sister with breastcancer onset at age of 40. Together, it suggests that GT198 activity isregulated through its alternative splicing variant. The increased splicevariant activity of the GT198 gene may be involved in cancer initiation.GT198 is a novel breast and ovarian cancer susceptibility genepotentially also contributing to 17q21-associated cancer predisposition.

GT198 is a small protein capable of forming a homo- and heterodimer andinteracting with DNA-binding proteins including the zinc finger domainsof nuclear receptors and Rad51. The dual function of GT198 intranscription and DNA repair mirrors that of BRCA1 and BRCA2. SinceBRCA1 contains a C-terminal domain directly modifying RNA polymerase IIin transcription (Bennett, C. B. et al., PLoS ONE, 3:e1448 (2008)), andhas been shown function as a nuclear receptor coactivator, thetranscriptional activity of GT198 may be mediated by BRCA1 when theywork in concert. In contrast, the hormone-induced activity of BRCA1 inbreast and ovarian cancers is potentially through nuclearreceptor-associated GT198. A plausible model for GT198 as a partner forBRCA1 would explain their coexpression in tissues, their coordinatedregulation in stem cell differentiation, their involvement in bothtranscription and DNA repair pathways, and their alteration in breastand ovarian cancers. Previous evidence supports the equal involvement ofboth steroid hormone regulation and DNA repair activity in breast andovarian cancer initiation.

Alternative splicing control is an integral step in pre-mRNAtranscription, and more than 90% of multi-exon genes in the human genomeare alternatively spliced (Pan, Q., et al., Nat Genet, 40:1413-5(2008)). Splicing variants influence wild type activities in normaldevelopment and cellular differentiation. Splicing defects arefrequently found in disease or cancer (Kalnina, Z., Genes ChromosomesCancer, 42:342-57 (2005); Venables, J. P., Bioessays, 28:378-86 (2006)).Mutation screening of BRCA1 and BRCA2 showed thousands of changes(Meindl, A., Int J Cancer, 97:472-80 (2002)), many of which may alteralternative splicing (Brose, M. S., et al., Genet Test, 8:133-8 (2004)).The allelic sequence loss and rearrangement at distant enhancers or atthe promoter region of BRCA1 may also affect its alternative splicingregulation (Orban, T. I. & Olah, E., Mol Pathol, 56:191-7 (2003)).Multiple splicing variants of GT198 lead to the same functionalconsequence. If this phenomenon is also present in BRCA1, the wild typeBRCA1 activity could be controlled by a variety of its variants.Differential expression of wild type and variant transcripts can beaccomplished via two transcriptional start sites, which alternate theusage during early stem cell differentiation and are similarly observedin GT198 and BRCA1. An increased expression of wild-type BRCA2 is alsofound during stem cell differentiation, although the BRCA2 variants areless well characterized. Consistently, the variant to wild type switchis present in the previously characterized oncogene CoAA suggesting theessential role of alternative splicing in stem cell differentiation(Brooks, Y. S., et al., J Biol Chem, 284:18033-46 (2009); Yang, Z., etal., Nucleic Acids Res, 35:1919-1932 (2007)). Since splicing forms arecompetitively expressed, and often functionally counteract, thedown-regulation of variants permits an up-regulation of wild types. Incontrast, aberrant up-regulation of variants, caused by a wide range ofmutations, could be phenotypically equivalent to its wild-typedeficiency, i.e., a “loss of tumor suppressor”. This will prevent normalcell differentiation when GT198 or BRCA1 is required.

Early evidence from genetic linkage studies demonstrated the closeassociation of familial breast cancers equal to BRCA1 and to HSD17B1markers (Hall, J. M., et al., Science, 250:1684-9 (1990); Hall, J. M.,et al., Am J Hum Genet, 50:1235-42 (1992); Anderson, L. A., et al.,Genomics, 17:618-23 (1993); Black, D. M., et al., Am J Hum Genet,52:702-10 (1993); Easton, D. F., Bishop, D. T., et al., Am J Hum Genet.52:678-701 (1993)), the latter encodes 17β-hydroxysteroid dehydrogenase.Since mutations were not identified within the HSD17B1 gene in linkedfamilies (Simard, J., et al., Hum Mol Genet, 2:1193-9 (1993)), theeffort was later focused on the BRCA1 marker. The cloning of BRCA1 wasbased on the linkage of the families to the D17S1321 and D17S1325 regionwhich flanks BRCA1 but excludes the HSD17B1 and GT198 genes (Mild, Y.,et al., Science, 266:66-71 (1994); Neuhausen, S. L., et al., Hum MolGenet, 3:1919-26 (1994)). GT198 has a small gene size at 5 Kb nearHSD17B1 and was thus missed in the historical candidate geneidentification. Subsequent studies showed limited involvement of BRCA1mutations in 17q21-associated cancer families leading to the speculationof additional unidentified candidate within the BRCA1 locus (Vogelstein,B. & Kinzler, K. W., Cell, 79:1-3 (1994)). In view of the functional andgenetic evidence of GT198 as described herein, GT198 is potentiallyanother breast cancer susceptibility gene at chromosome 17q21 locus.

There is another possible reason for GT198 not being geneticallyidentified in past investigations of sporadic breast and ovariancancers. Most genetic analyses for somatic mutations relied on tumormass but not on rare cancer-initiating cells. One emerging hypothesis,still under debate, is that tumor cells do not grow out ofcancer-initiating cells carrying first hit genetic mutation. Instead,tumor growth is influenced by tumor environments containingcancer-initiating cells. If this hypothesis proves true, the geneticalterations in a small percentage of GT198 positive cells are unlikelyto be identified from the tumor mass without a visible marker. Thus, thefunctional analysis comparison of GT198 and BRCA1 might be an essentialstep towards identifying this candidate that could also be involved insporadic cancers as supported by its expression patterns in primarytumors.

B. Diagnostics

GT198 variant proteins and nucleic acids encoding them or fragmentsthereof, can be used in diagnostic assays, screening assays, and intherapeutic applications. In some embodiments, the compositions are usedas diagnostic markers for the detection of cancer due to expression ofGT198 variants, preferably alternatively spliced variants. Exemplaryvariants include GT198a, GT198-1, GT198-2, GT198-3, GT198-4, andGT198a-4. Representative cancers include, but are not limited to ovarianand breast cancer. Detection of elevated levels of expression of one ormore GT198 variants in tissue or subjects allows for a determination ordiagnosis of cancer such as breast and ovarian cancers. The GT198 can bea polypeptide or nucleic acid. To detect or diagnose cancer, baselinevalues for the expression or activity of GT198 can be established toprovide a basis for the diagnosis and/or prognosis of cancer in asubject. Preferred subjects include, mammals including but not limitedto humans. In some embodiments, this is accomplished by combining bodyfluids, tissue biopsies, or cell extracts taken from normal subjects(cancer-free subjects) with one or more antibody(ies) to or nucleicacids that specifically hybridize to a nucleic acid encoding a GT198variant under conditions suitable for complex formation. Such conditionsare well known in the art. The amount of standard complex formation maybe quantified by comparing levels of antibody-target complex in thenormal sample with a dilution series of positive controls, in which aknown amount of antibody is combined with known concentrations ofpurified GT198 variant. Standard values obtained from normal samples maybe compared with values obtained from samples from subjects suspected ofhaving cancer. Deviation between standard and subject values establishesthe presence of or predisposition to the disease state.

In other embodiments, the expression levels of GT198 splice variants aredetermined for different cellular states in the cancer phenotype; thatis, the expression levels of GT198 variants in cancer-free tissue and incancer tissue are evaluated to provide expression profiles. Anexpression profile of a particular cell state or point of development isessentially a “fingerprint” of the state; while two states may have anyparticular gene or GT198 variant similarly expressed, the evaluation ofa number of genes or GT198 variants simultaneously allows the generationof a gene or GT198 variant expression profile that is unique to thestate of the cell. By comparing expression profiles of cells indifferent states, information regarding which genes or GT198 variantsare important (including both up- and down-regulation of genes orvariants) in each of these states is obtained. Then, diagnosis may bedone or confirmed by determining whether or not the tissue from aparticular patient has the gene expression profile of normal orcancerous tissue.

“Differential expression,” or grammatical equivalents as used herein,refers to both qualitative as well as quantitative differences in theGT198 variant's temporal and/or cellular expression patterns within andamong the cells. Thus, a differentially expressed GT198 variant canqualitatively have its expression altered, including an activation orinactivation, in, for example, normal versus ovarian cancer tissue. Thatis, GT198 variants may be turned on or turned off in a particular state,relative to another state. As is apparent to the skilled artisan, anycomparison of two or more states can be made. Such a qualitativelyregulated GT198 or variant thereof will exhibit an expression patternwithin a state or cell type which is detectable by standard techniquesin one such state or cell type, but is not detectable in both.Alternatively, the determination is quantitative in that expression isincreased or decreased; that is, the expression of the GT198 variant iseither upregulated, resulting in an increased amount of transcript, ordownregulated, resulting in a decreased amount of transcript. The degreeto which expression differs need only be large enough to quantify viastandard characterization techniques as outlined below, such as by useof Affymetrix GeneChip™ expression arrays, Lockhart, NatureBiotechnology, 14:1675-1680 (1996). Other techniques include, but arenot limited to, quantitative reverse transcriptase PCR, Northernanalysis and RNase protection. The change in expression (i.e.,upregulation or downregulation) is at least about 50%, more preferablyat least about 100%, more preferably at least about 150%, morepreferably, at least about 200%, with from 300 to at least 1000% beingespecially preferred.

As will be appreciated by those in the art, this may be done byevaluation at either the GT198 variant transcript, or the protein level;that is, the amount of gene expression may be monitored using nucleicacid probes to the DNA or RNA equivalent of the GT198 varianttranscript, and the quantification of GT198 variant expression levels,or, alternatively, the final GT198 variant product itself (protein) canbe monitored, for example through the use of antibodies to the GT198variant protein and standard immunoassays (ELISAs, etc.) or othertechniques, including mass spectroscopy assays, 2D gel electrophoresisassays, etc. Thus, the proteins corresponding to the GT198 variantsi.e., those identified as being important in a cancer phenotype, can beevaluated in a cancer diagnostic test.

In some embodiments, antibodies to the GT198 variant can be used in insitu imaging techniques. Preferably the antibody is specific to theGT198 variant polypeptide and does not shown detectable binding to thewild-type GT198 polypeptide. In this method cells are contacted withfrom one to many antibodies to a GT198 variant polypeptide. Followingwashing to remove non-specific antibody binding, the presence of theantibody or antibodies is detected. In one embodiment the antibody isdetected by incubating with a secondary antibody that contains adetectable label. In another method the primary antibody to the GT198variant contains a detectable label. In another preferred embodimenteach one of multiple primary antibodies contains a distinct anddetectable label. This method finds particular use in simultaneousscreening for a plurality of cancer markers, for example BRCA1 or BRCA2.As will be appreciated by one of ordinary skill in the art, numerousother histological imaging techniques can be used.

In some embodiments the label is detected in a fluorometer which has theability to detect and distinguish emissions of different wavelengths. Inaddition, a fluorescence activated cell sorter (FACS) can be used in themethod.

In some embodiments, in situ hybridization of labeled GT198 or GT198nucleic acid probes to tissue arrays is done. For example, arrays oftissue samples, including cancer tissue and/or normal (cancer-freetissue), are made. In situ hybridization as is known in the art can thenbe done. Cells having elevated levels of one or more GT198 variantsrelative to a control are indicative of cancer. An exemplary controlincludes cells from a subject without cancer.

It is understood that when comparing the expression fingerprints betweenan individual and a standard, the skilled artisan can make a diagnosisas well as a prognosis. It is further understood that the genes whichindicate the diagnosis may differ from those which indicate theprognosis. The data from the disclosed assays can be used to assist inthe diagnosis of cancer.

In a preferred embodiment, the GT198 variant proteins, antibodies,nucleic acids are used in prognosis assays. In some embodiments, geneexpression profiles can be generated that correlate to cancer severity,in terms of long term prognosis. Again, this may be done on either aprotein or gene level, with the use of genes being preferred. In someembodiments, GT198 proteins or nucleic acid probes are attached to solidsupports for the detection and quantification of GT198 variant sequencesin a tissue or subject. The assays proceed as outlined for diagnosis.

In one embodiment, the presence of wild-type GT198 in the cytosol ofcells is indicative of cancer. Typically, antibodies specific towild-type GT198 can be used in immunological assays of cells or tissueto detect or quantify GT198 in the cytosol. In another embodiment,elevated levels of cytoplasmic expression of wild-type GT198 relative toa control is indicative of cancer.

Another embodiment provides a method for assisting in the diagnosis ofcancer by determining expression of GT198 variants in a sample obtainedfrom a subject in combination or alternation with determining theexpression of mutations in one or more additional genes. For example,the method can include determining the expression of mutations in BRCA1or BRCA2 as well as determining the expression of one or more GT198variants.

C. Efficacy of Therapeutic Agents

The efficacy of therapeutic agents, such as antibodies and/or othercandidate drugs also can be determined using the diagnostic assaysdescribed above. As will be appreciated by a person of skill in the art,assays to determine the efficacy of a therapeutic agent require theestablishment of baseline values. In some embodiments, this isaccomplished by combining body fluids, tissue biopsies, or cell extractstaken from a subject with cancer prior to treatment with the candidatedrug with one or more antibody(ies) to a GT198 variant under conditionssuitable for complex formation. Such conditions are well known in theart. The amount of standard complex formation may be quantified bycomparing levels of antibody-target complex in the normal sample with adilution series of positive controls, in which a known amount ofantibody is combined with known concentrations of purified GT198variant. Standard values obtained from a patient before treatment may becompared with values obtained from a subject after treatment. Deviationbetween standard and subject values establishes the efficacy of thedrug.

II. Screening Assays

In some embodiments, the GT198 variant proteins, antibodies, nucleicacids, and cells containing the GT198 variant proteins or nucleic acidsare used in screening assays. For example, screens for agents thatmodulate the cancer phenotype can be run. This can be done by screeningfor modulators of gene expression including the expression of GT198variants or for modulators of GT198 variant protein activity at theindividual gene or protein level or by evaluating the effect of drugcandidates on a “gene expression profile”. In some embodiments, theexpression profiles are used in conjunction with high throughputscreening techniques to allow monitoring for expression profile genesafter treatment with a candidate agent (see Zlokarnik, et al., Science,279:84-8 (1998)).

“Modulation” includes both an increase and a decrease in gene expressionor activity. The preferred amount of modulation will depend on theoriginal change of the gene expression in normal versus tumor tissue,with changes of at least 10%, preferably 50%, more preferably 100-300%,and in some embodiments 300-1000% or greater. If a gene exhibits a 4fold increase in tumor compared to normal tissue, a decrease of aboutfour fold is desired; a 10 fold decrease in tumor compared to normaltissue gives a 10 fold increase in expression for a candidate agent isdesired, etc.

As will be appreciated by those in the art, this may be done byevaluation at either the variant transcript or the protein level; thatis, the amount of GT198 variant expression may be monitored usingnucleic acid probes and the quantification of mRNA expression levels,or, alternatively, the level of the gene product itself can bemonitored, for example through the use of antibodies to the GT198variant and standard immunoassays. Alternatively, binding andbioactivity assays with the protein may be done as outlined below.

In some embodiments, gene expression monitoring is done and a number ofgenes in addition to GT198 variants, i.e. an expression profile, aremonitored simultaneously. If desired, multiple protein expressionmonitoring can be done as well. In embodiments monitoring multiple genesor proteins, the corresponding GT198 variant probes are immobilized tosolid supports. It is understood that immobilization can occur by anymeans, including for example; by covalent attachment, by electrostaticimmobilization, by attachment through a ligand/ligand interaction, bycontact or by depositing on the surface. “Solid support” or “solidsubstrate” refers to any solid phase material upon which a GT198 variantsequence, or antibody is synthesized, attached, ligated or otherwiseimmobilized. A solid support may be composed of organic polymers such aspolystyrene, polyethylene, polypropylene, polyfluoroethylene,polyethyleneoxy, and polyacrylamide, as well as co-polymers and graftsthereof. A solid support may also be inorganic, such as glass, silica,controlled-pore-glass (CPG), or reverse-phase silica. The configurationof a solid support may be in the form of beads, spheres, particles,granules, a gel, or a surface. Surfaces may be planar, substantiallyplanar, or non-planar. Solid supports may be porous or non-porous, andmay have swelling or non-swelling characteristics. A solid support maybe configured in the form of a well, depression or other container,vessel, feature or location. A plurality of solid supports may beconfigured in an array at various locations, addressable for roboticdelivery of reagents, or by detection means including scanning by laserillumination and confocal or deflective light gathering.

Generally, a candidate bioactive agent is added prior to analysis. Theterm “candidate bioactive agent” or “drug candidate” or grammaticalequivalents as used herein describes any molecule, e.g., protein,oligopeptide, small organic or inorganic molecule, polysaccharide,polynucleotide, etc., to be tested for bioactive agents that are capableof directly or indirectly altering either the cancer phenotype, bindingto and/or modulating the bioactivity of a GT198 variant, or theexpression of a GT198 variant sequence. In a particularly preferredembodiment, the candidate agent suppresses the cancer phenotype, forexample to a normal tissue fingerprint. Generally a plurality of assaymixtures is run in parallel with different agent concentrations toobtain a differential response to the various concentrations. Typically,one of these concentrations serves as a negative control, i.e., at zeroconcentration or below the level of detection. In a preferredembodiment, the expression of one or more GT198 variant is inhibited.

In one aspect, a candidate agent will neutralize the effect of one ormore GT198 variant. By “neutralize” it is meant that activity of aprotein is either inhibited or counter-acted against so as to havesubstantially no effect on a cell.

Candidate agents encompass numerous chemical classes, though typicallythey are organic or inorganic molecules, preferably small organiccompounds having a molecular weight of more than 100 and less than about2,500 daltons. Preferred small molecules are less than 2000, or lessthan 1500 or less than 1000 or less than 500 D. Candidate agents includefunctional groups necessary for structural interaction with proteins,particularly hydrogen bonding, and typically include at least an amine,carbonyl, hydroxyl or carboxyl group, preferably at least two of thefunctional chemical groups. The candidate agents often comprise cyclicalcarbon or heterocyclic structures and/or aromatic or polyaromaticstructures substituted with one or more of the above functional groups.Candidate agents are also found among biomolecules including peptides,proteins, saccharides, fatty acids, steroids, purines, pyrimidines,derivatives, structural analogs or combinations thereof.

Candidate agents are obtained from a wide variety of sources includinglibraries of synthetic or natural compounds. For example, numerous meansare available for random and directed synthesis of a wide variety oforganic compounds and biomolecules, including expression of randomizedoligonucleotides. Alternatively, libraries of natural compounds in theform of bacterial, fungal, plant and animal extracts are available orreadily produced. Additionally, natural or synthetically producedlibraries and compounds are readily modified through conventionalchemical, physical and biochemical means. Known pharmacological agentsmay be subjected to directed or random chemical modifications, such asacylation, alkylation, esterification, amidification to producestructural analogs.

In assays for altering the expression profile of one or more GT198variant sequences, after the candidate agent has been added and thecells allowed to incubate for some period of time, the sample containingthe GT198 variant sequences to be analyzed is added to a solid support.If required, the GT198 variant sequence is prepared using knowntechniques. For example, the sample may be treated to lyse the cells,using known lysis buffers, electroporation, etc., with purificationand/or amplification such as PCR occurring as needed, as will beappreciated by those in the art.

Generally, one of the assay components is labeled to provide a means ofdetecting the binding complex of interest. By “labeled” herein is meantthat a compound has at least one element, isotope or chemical compoundattached to enable the detection of the compound. In general, labelsfall into three classes: a) isotopic labels, which may be radioactive orheavy isotopes; b) immune labels, which may be antibodies or antigens;and c) colored or fluorescent dyes. The labels may be incorporated intothe GT198 variant nucleic acids, proteins and antibodies at anyposition. For example, the label should be capable of producing, eitherdirectly or indirectly, a detectable signal. The detectable moiety maybe a radioisotope, such as ³H, ¹⁴C, ³²P, ³⁵S, or ¹²⁵I, a fluorescent orchemiluminescent compound, such as fluorescein isothiocyanate,rhodamine, or luciferin, or an enzyme, such as alkaline phosphatase,beta-galactosidase or horseradish peroxidase. Any method known in theart for conjugating the antibody to the label may be employed, includingthose methods described by Hunter et al., Nature, 144:945 (1962); Davidet al., Biochemistry, 13:1014 (1974); Pain et al., J. Immunol. Meth.,40:219 (1981); and Nygren, J. Histochem. and Cytochem., 30:407 (1982)).The label also can be an enzyme, such as, alkaline phosphatase orhorseradish peroxidase, which when provided with an appropriatesubstrate produces a product that can be detected. Alternatively, thelabel can be a labeled compound or small molecule, such as an enzymeinhibitor, that binds but is not catalyzed or altered by the enzyme. Thelabel also can be a moiety or compound, such as, an epitope tag orbiotin which specifically binds to streptavidin. For the example ofbiotin, the streptavidin is labeled as described above, thereby,providing a detectable signal for the bound target sequence. As known inthe art, unbound labeled streptavidin is removed prior to analysis.

As will be appreciated by those in the art, these assays can be directhybridization assays or can include “sandwich assays”, which include theuse of multiple probes, as is generally outlined in U.S. Pat. Nos.5,681,702, 5,597,909, 5,545,730, 5,594,117, 5,591,584, 5,571,670,5,580,731, 5,571,670, 5,591,584, 5,624,802, 5,635,352, 5,594,118,5,359,100, 5,124,246 and 5,681,697, all of which are hereby incorporatedby reference in their entirety.

A variety of hybridization conditions may be used, including high,moderate and low stringency. The assays are generally run understringency conditions which allows formation of the label probehybridization complex only in the presence of target. Stringency can becontrolled by altering a step parameter that is a thermodynamicvariable, including, but not limited to, temperature, formamideconcentration, salt concentration, chaotropic salt concentration pH,organic solvent concentration, etc. To achieve specific hybridizationunder a variety of conditions, probes specific for GT198 variantpolynucleotides are used. The probe is less than about 1000 nucleotidesin length, preferably less than 500 nucleotides in length. Preferredprobes are preferably at least about 10 nucleotides in length, and mostpreferably at least about 20 nucleotides in length. Such probes may beused to amplify corresponding GT198 variant sequences from a sample byPCR.

Hybridization may be carried out under stringent conditions. By“stringent conditions” or “stringent hybridization conditions” isintended conditions under which a probe will hybridize to its targetsequence to a detectably greater degree than to other sequences (e.g.,at least 2-fold over background). Stringent conditions aresequence-dependent and will be different in different circumstances. Bycontrolling the stringency of the hybridization and/or washingconditions, target sequences that are 100% complementary to the probecan be identified (homologous probing). Alternatively, stringencyconditions can be adjusted to allow some mismatching in sequences sothat lower degrees of similarity are detected (heterologous probing).

Typically, stringent conditions will be those in which the saltconcentration is less than about 1.5 M Na ion, typically about 0.01 to1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and thetemperature is at least about 30° C. for short probes (e.g., 10 to 50nucleotides) and at least about 60° C. for long probes (e.g., greaterthan 50 nucleotides). Stringent conditions may also be achieved with theaddition of destabilizing agents such as formamide. Exemplary lowstringency conditions include hybridization with a buffer solution of 30to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulphate) at 37° C.,and a wash in 1× to 2× standard sodium citrate (SSC) (20×SSC=3.0 M NaCl0.3 M trisodium citrate) at 50 to 55° C. Exemplary moderate stringencyconditions include hybridization in 40 to 45% formamide, 1.0 M NaCl, 1%SDS at 37° C., and a wash in 0.5× to 1×SSC at 55 to 60° C. Exemplaryhigh stringency conditions include hybridization in 50% formamide, 1 MNaCl, 1% SDS at 37° C., and a wash in 0.1×SSC at 60 to 65° C.Optionally, wash buffers may include about 0.1% to about 1% SDS.Duration of hybridization is generally less than about 24 hours, usuallyabout 4 to about 12 hours.

These parameters may also be used to control non-specific binding, as isgenerally outlined in U.S. Pat. No. 5,681,697. Thus it may be desirableto perform certain steps at higher stringency conditions to reducenon-specific binding.

The reactions outlined herein may be accomplished in a variety of ways,as will be appreciated by those in the art. Components of the reactionmay be added simultaneously, or sequentially, in any order, withpreferred embodiments outlined below. In addition, the reaction mayinclude a variety of other reagents may be included in the assays. Theseinclude reagents like salts, buffers, neutral proteins, e.g. albumin,detergents, etc which may be used to facilitate optimal hybridizationand detection, and/or reduce non-specific or background interactions.Also reagents that otherwise improve the efficiency of the assay, suchas protease inhibitors, nuclease inhibitors, anti-microbial agents,etc., may be used, depending on the sample preparation methods andpurity of the target. In addition, either solid phase or solution based(i.e., kinetic PCR) assays may be used.

Once the assay is run, the data is analyzed to determine the expressionlevels, and changes in expression levels as between states, of GT198variants, or individual GT198 variant proteins, forming an expressionprofile.

In some embodiments, screening is done to alter the biological functionof the expression product of the GT198 variant. Again, having identifiedthe importance of a variant in a particular state, screening for agentsthat bind and/or modulate the biological activity of the variant can berun as is more fully outlined below.

In some embodiments, screens are designed to first find candidate agentsthat can bind to GT198 variant proteins or nucleic acids, and then theseagents may be used in assays that evaluate the ability of the candidateagent to modulate the GT198 variant activity and the cancer phenotype.As will be appreciated by those in the art, there are a number ofdifferent assays which may be run; binding assays and activity assays.

In some embodiments, binding assays are done. In general, purified orisolated GT198 variant proteins or nucleic acids are used. The methodsinclude combining a GT198 protein or nucleic acids and a candidatebioactive agent, and determining the binding of the candidate agent tothe GT198 variant protein or nucleic acids. Generally, the GT198 variantprotein or nucleic acids or the candidate agent is non-diffusably boundto a solid support having isolated sample receiving areas (e.g., amicrotiter plate, an array, etc.). Microtiter plates and arrays areespecially convenient because a large number of assays can be carriedout simultaneously, using small amounts of reagents and samples. Theparticular manner of binding of the composition is not crucial so longas it is compatible with the reagents, maintains the activity of thecomposition and is nondiffusable. Preferred methods of binding includethe use of antibodies (which do not sterically block either the ligandbinding site or activation sequence when the protein is bound to thesupport), direct binding to “sticky” or ionic supports, chemicalcrosslinking, the synthesis of the protein or agent on the surface, etc.Following binding of the protein or agent, excess unbound material isremoved by washing. The sample receiving areas may then be blockedthrough incubation with bovine serum albumin (BSA), casein or otherinnocuous protein or other moiety.

In some embodiments, the GT198 variant protein or nucleic acids arebound to the support, and a candidate bioactive agent is added to theassay. Alternatively, the candidate agent is bound to the support andthe GT198 variant protein or nucleic acids are added. Novel bindingagents include specific antibodies, non-natural binding agentsidentified in screens of chemical libraries, peptide analogs, aptamers,etc. Of particular interest are screening assays for agents that have alow toxicity for human cells. A wide variety of assays may be used forthis purpose, including labeled in vitro protein-protein binding assays,electrophoretic mobility shift assays, immunoassays for protein binding,functional assays (phosphorylation assays, etc.) and the like.

The determination of the binding of the candidate bioactive agent to theGT198 variant protein or nucleic acids may be done in a number of ways.In a preferred embodiment, the candidate bioactive agent is labeled, andbinding determined directly. For example, this may be done by attachingall or a portion of the GT198 variant protein or nucleic acids to asolid support, adding a labeled candidate agent (for example afluorescent label), washing off excess reagent, and determining whetherthe label is present on the solid support. Various blocking and washingsteps may be utilized as is known in the art.

In some embodiments, only one of the components is labeled. For example,the proteins (or proteinaceous candidate agents) may be labeled attyrosine positions using ¹²⁵I, or with fluorophores. Alternatively, morethan one component may be labeled with different labels; using ¹²⁵I forthe proteins, for example, and a fluorophor for the candidate agents.

In some embodiments, the binding of the candidate bioactive agent isdetermined through the use of competitive binding assays. In thisembodiment, the competitor is a binding moiety known to bind to theGT198 variant protein or nucleic acid, such as an antibody, peptide,binding partner, ligand, etc. Under certain circumstances, there may becompetitive binding as between the bioactive agent and the bindingmoiety, with the binding moiety displacing the bioactive agent.

In some embodiments, the candidate bioactive agent is labeled. Eitherthe candidate bioactive agent, or the competitor, or both, is addedfirst to the protein for a time sufficient to allow binding, if present.Incubations may be performed at any temperature which facilitatesoptimal activity, typically between 4 and 40° C. Incubation periods areselected for optimum activity, but may also be optimized to facilitaterapid high through put screening. Typically between 0.1 and 1 hour willbe sufficient. Excess reagent is generally removed or washed away. Thesecond component is then added, and the presence or absence of thelabeled component is followed, to indicate binding.

In some embodiments, the competitor is added first, followed by thecandidate bioactive agent. Displacement of the competitor is anindication that the candidate bioactive agent is binding to the GT198variant protein or nucleic acid and thus is capable of binding to, andpotentially modulating, the activity of GT198 variant protein or nucleicacid. In this embodiment, either component can be labeled. Thus, forexample, if the competitor is labeled, the presence of label in the washsolution indicates displacement by the agent. Alternatively, if thecandidate bioactive agent is labeled, the presence of the label on thesupport indicates displacement.

In other embodiments, the candidate bioactive agent is added first, withincubation and washing, followed by the competitor. The absence ofbinding by the competitor may indicate that the bioactive agent is boundto the GT198 variant protein or nucleic acid with a higher affinity.Thus, if the candidate bioactive agent is labeled, the presence of thelabel on the support, coupled with a lack of competitor binding, mayindicate that the candidate agent is capable of binding to the GT198variant protein or nucleic acid.

In some embodiments, the methods include differential screening toidentity bioactive agents that are capable of modulating the activity ofthe GT198 variant protein or nucleic acid. In this embodiment, themethods include combining a GT198 variant protein or nucleic acid and acompetitor in a first sample. A second sample comprises a candidatebioactive agent, a GT198 variant protein or nucleic acid and acompetitor. The binding of the competitor is determined for bothsamples, and a change, or difference in binding between the two samplesindicates the presence of an agent capable of binding to the GT198variant protein or nucleic acid and potentially modulating its activity.That is, if the binding of the competitor is different in the secondsample relative to the first sample, the agent is capable of binding tothe GT198 variant protein or nucleic acid.

In some embodiments, methods for screening for bioactive agents capableof modulating the activity of a GT198 variant protein or nucleic acid ina cell are provided. The methods include adding a candidate bioactiveagent, as defined above, to a cell having GT198 variant protein ornucleic acid. Typically, cells having one or more amplicons of GT198variant are used. Methods for culturing cells and for assaying cellscattering, adhesion and migration are described in Russell et al., J.Cell Sci., 116:3543-3556 (2003), the entire contents of which areincorporated herein by reference.

Positive controls and negative controls may be used in the assays.Preferably all control and test samples are performed in at leasttriplicate to obtain statistically significant results. Incubation ofall samples is for a time sufficient for the binding of the agent to theprotein. Following incubation, all samples are washed free ofnon-specifically bound material and the amount of bound, generallylabeled agent determined. For example, where a radiolabel is employed,the samples may be counted in a scintillation counter to determine theamount of bound compound.

A variety of other reagents may be included in the screening assays.These include reagents like salts, neutral proteins, e.g. albumin,detergents, etc which may be used to facilitate optimal protein-proteinbinding and/or reduce non-specific or background interactions. Alsoreagents that otherwise improve the efficiency of the assay, such asprotease inhibitors, nuclease inhibitors, anti-microbial agents, etc.,may be used. The mixture of components may be added in any order thatprovides for the requisite binding.

In one aspect, the assays are evaluated in the presence or absence orprevious or subsequent exposure of physiological signals, for examplehormones, antibodies, peptides, antigens, cytokines, growth factors,action potentials, pharmacological agents including chemotherapeutics,radiation, carcinogenics, or other cells (i.e. cell-cell contacts). Inanother example, the determinations are determined at different stagesof the cell cycle process.

III. Pharmaceutical Compositions and Methods of Treatment

A. Pharmaceutical Compositions

Another embodiment provides pharmaceutical compositions containing oneor more of antagonists of a GT198 variant protein or nucleic acid. By“pharmacological activity” herein is meant that the compounds are ableto inhibit or interfere with the activity of GT198 variant protein ornucleic acid. The compounds having the desired pharmacological activitymay be administered in a physiologically acceptable carrier to a subjector patient. A “subject” or “patient” includes both humans and otheranimals, particularly mammals, and domestic animals. Thus, the methodsare applicable to both human therapy and veterinary applications.

In some embodiments, bioactive agents include antibodies that recognizeGT198 variant protein and that have been demonstrated to inhibit ormodulate GT198 variant protein activity or bioavailability. In otherembodiments, bioactive agents include antisense or siRNA compositionsagainst GT198 intron 1 sequence:gtaacggcgccgtgggcgcggggaagacccgggagggcagtgggtgagGaggtcggttgagtggccccctcccctgcctttctctccgtag (SEQ ID NO: 4). These agentscan be delivered directly or in pharmaceutical compositions along withsuitable carriers or excipients, as well known in the art. Presentmethods of treatment include embodiments providing for administration ofan effective amount of a compound or agent that inhibits the activity orexpression of GT198 variant to a patient in need of treatment.

An effective amount of such agents can readily be determined by routineexperimentation, as can the most effective and convenient route ofadministration and the most appropriate formulation. Variousformulations and drug delivery systems are available in the art.

Suitable routes of administration may, for example, include oral,rectal, transmucosal, transdermal, nasal, or intestinal administrationand parenteral delivery, including intramuscular, subcutaneous,intramedullary injections, as well as intrathecal, directintraventricular, intravenous, intraperitoneal, intranasal, orintraocular injections. The agent or composition thereof may beadministered in a local rather than a systemic manner. For example, asuitable agent can be delivered via injection or in a targeted drugdelivery system, such as a depot or sustained release formulation.

The pharmaceutical compositions may be manufactured by any of themethods well-known in the art, such as by conventional mixing,dissolving, granulating, dragee-making, levigating, emulsifying,encapsulating, entrapping, or lyophilizing processes. The compositionscan include one or more physiologically acceptable carriers such asexcipients and auxiliaries that facilitate processing of activemolecules into preparations for pharmaceutical use. Proper formulationis dependent upon the route of administration chosen.

For example, for injection, the composition may be formulated in aqueoussolutions, preferably in physiologically compatible buffers such asHanks's solution, Ringer's solution, or physiological saline buffer. Fortransmucosal or nasal administration, penetrants appropriate to thebarrier to be permeated are used in the formulation. Such penetrants aregenerally known in the art. For oral administration, the agents can beformulated readily by combining the active agents with pharmaceuticallyacceptable carriers well known in the art. Such carriers enable theagents of the invention to be formulated as tablets, pills, dragees,capsules, liquids, gels, syrups, slurries, suspensions and the like, fororal ingestion by a subject. The agents may also be formulated in rectalcompositions such as suppositories or retention enemas, e.g., containingconventional suppository bases such as cocoa butter or other glycerides.

Pharmaceutical preparations for oral use can be obtained as solidexcipients, optionally grinding a resulting mixture, and processing themixture of granules, after adding suitable auxiliaries, if desired, toobtain tablets or dragee cores. Suitable excipients are, in particular,fillers such as sugars, including lactose, sucrose, mannitol, orsorbitol; cellulose preparations such as, for example, maize starch,wheat starch, rice starch, potato starch, gelatin, gum tragacanth,methyl cellulose, hydroxypropylmethyl-cellulose, sodiumcarboxymethylcellulose, and/or polyvinylpyrrolidone (PVP). If desired,disintegrating agents may be added, such as the cross-linked polyvinylpyrrolidone, agar, or alginic acid or a salt thereof such as sodiumalginate.

Dragee cores are provided with suitable coatings. For this purpose,concentrated sugar solutions may be used, which may optionally containgum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethyleneglycol, and/or titanium dioxide, lacquer solutions, and suitable organicsolvents or solvent mixtures. Dyestuffs or pigments may be added to thetablets or dragee coatings for identification or to characterizedifferent combinations of active agent doses.

Pharmaceutical preparations for oral administration include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules can contain the active ingredients in admixture with fillersuch as lactose, binders such as starches, and/or lubricants such astalc or magnesium stearate and, optionally, stabilizers. In softcapsules, the active agents may be dissolved or suspended in suitableliquids, such as fatty oils, liquid paraffin, or liquid polyethyleneglycols. In addition, stabilizers may be added. All formulations fororal administration should be in dosages suitable for suchadministration.

For administration by inhalation, the agents can be convenientlydelivered in the form of an aerosol spray presentation from pressurizedpacks or a nebuliser, with the use of a suitable propellant, e.g.,dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, carbon dioxide, or any other suitable gas. Inthe case of a pressurized aerosol, the appropriate dosage unit may bedetermined by providing a valve to deliver a metered amount. Capsulesand cartridges for use in an inhaler or insufflator may be formulated.These typically contain a powder mix of the agent and a suitable powderbase such as lactose or starch.

Compositions formulated for parenteral administration by injection,e.g., by bolus injection or continuous infusion can be presented in unitdosage form, e.g. in ampoules or in multi-dose containers, with an addedpreservative. The compositions may take such forms as suspensions,solutions or emulsions in oily or aqueous vehicles, and may containformulatory agents such as suspending, stabilizing and/or dispersingagents. Formulations for parenteral administration include aqueoussolutions of the compound or agent to be administered, including inwater-soluble form.

Suspensions of the active agents may also be prepared as appropriateoily injection suspensions. Suitable lipophilic solvents or vehiclesinclude fatty oils such as sesame oil and synthetic fatty acid esters,such as ethyl oleate or triglycerides, or liposomes. Aqueous injectionsuspensions may contain substances that increase the viscosity of thesuspension, such as sodium carboxymethyl cellulose, sorbitol, ordextran. Optionally, the suspension may also contain suitablestabilizers or agents that increase the solubility of the agents toallow for the preparation of highly concentrated solutions.Alternatively, the active ingredient may be in powder form forconstitution with a suitable vehicle, e.g., sterile pyrogen-free water,before use.

As mentioned above, the compositions can also be formulated as a depotpreparation. Such long acting formulations may be administered byimplantation (for example, subcutaneously or intramuscularly) or byintramuscular injection. Thus, for example, the present agents may beformulated with suitable polymeric or hydrophobic materials (for exampleas an emulsion in an acceptable oil) or ion exchange resins, or assparingly soluble derivatives, for example, as a sparingly soluble salt.

Suitable carriers for the hydrophobic molecules of the invention arewell-known in the art and include co-solvent systems comprising, forexample, benzyl alcohol, a nonpolar surfactant, a water-miscible organicpolymer, and an aqueous phase. The co-solvent system may be the VPDco-solvent system. VPD is a solution of 3% w/v benzyl alcohol, 8% w/v ofthe nonpolar surfactant polysorbate 80, and 65% w/v polyethylene glycol300, made up to volume in absolute ethanol. The VPD co-solvent system(VPD: 5 W) consists of VPD diluted 1:1 with a 5% dextrose in watersolution. This co-solvent system is effective in dissolving hydrophobicagents and produces low toxicity upon systemic administration.Naturally, the proportions of a co-solvent system may be variedconsiderably without destroying its solubility and toxicitycharacteristics. Furthermore, the identity of the co-solvent componentsmay be varied. For example, other low-toxicity nonpolar surfactants maybe used instead of polysorbate 80, the fraction size of polyethyleneglycol may be varied, other biocompatible polymers may replacepolyethylene glycol, e.g. polyvinyl pyrrolidone, and other sugars orpolysaccharides may substitute for dextrose.

Alternatively, other delivery systems for hydrophobic molecules may beemployed. Liposomes and emulsions are well known examples of deliveryvehicles or carriers for hydrophobic drugs. Liposomal delivery systemsare discussed above in the context of gene-delivery systems. Certainorganic solvents such as dimethylsulfoxide also may be employed,although usually at the cost of greater toxicity. Additionally, theagents may be delivered using sustained-release systems, such assemi-permeable matrices of solid hydrophobic polymers containing theeffective amount of the composition to be administered. Varioussustained-release materials are established and available to those ofskill in the art. Sustained-release capsules may, depending on theirchemical nature, release the agents for a few weeks up to over 100 days.Depending on the chemical nature and the biological stability of thetherapeutic reagent, additional strategies for protein stabilization maybe employed.

For any composition employed herein, a therapeutically effective dosecan be estimated initially using a variety of techniques well-known inthe art. For example, in a cell culture assay, a dose can be formulatedin animal models to achieve a circulating concentration range thatincludes the IC₅₀ as determined in cell culture. Where inhibition ofGT198 or GT198 variant activity is desired, the concentration of thetest agent that achieves a half-maximal inhibition of GT198 or GT198variant activity can be determined. Dosage ranges appropriate for humansubjects can be determined, using data obtained from cell culture assaysand other animal studies.

A therapeutically effective dose of an agent refers to that amount ofthe agent that results in amelioration of symptoms or a prolongation ofsurvival in a subject. Toxicity and therapeutic efficacy of suchmolecules can be determined by standard pharmaceutical procedures incell cultures or experimental animals, e.g., by determining the LD₅₀(the dose lethal to 50% of the population) and the ED₅₀ (the dosetherapeutically effective in 50% of the population). The dose ratio oftoxic to therapeutic effects is the therapeutic index, which can beexpressed as the ratio LD₅₀/ED₅₀. Agents that exhibit high therapeuticindices are preferred.

Dosages preferably fall within a range of circulating concentrationsthat includes the ED₅₀ with little or no toxicity. Dosages may varywithin this range depending upon the dosage form employed and the routeof administration utilized. The exact formulation, route ofadministration, and dosage should be chosen, according to methods knownin the art, in view of the specifics of a subject's condition.

Dosage amount and interval may be adjusted individually to provideplasma levels or tissue levels of the active moiety which are sufficientto affect the expression or activity of GT198 or GT198 variant, asdesired, i.e. minimal effective concentration (MEC). The MEC will varyfor each agent but can be estimated from, for example, in vitro data,such as the concentration necessary to achieve 50-90% inhibition ofGT198 or GT198 variant activity using the assays described herein.Dosages necessary to achieve the MEC will depend on individualcharacteristics and route of administration. Agents or compositionsthereof should be administered using a regimen which maintains plasmalevels above the MEC for about 10-90% of the duration of treatment,preferably about 30-90% of the duration of treatment, and mostpreferably between 50-90%. In cases of local administration or selectiveuptake, the effective local concentration of the drug may not be relatedto plasma concentration.

The amount of agent or composition administered will, of course, bedependent on a variety of factors, including the sex, age, and weight ofthe subject being treated, the severity of the affliction, the manner ofadministration, and the judgment of the prescribing physician.

The present compositions may, if desired, be presented in a pack ordispenser device containing one or more unit dosage forms containing theactive ingredient. Such a pack or device may, for example, comprisemetal or plastic foil, such as a blister pack. The pack or dispenserdevice may be accompanied by instructions for administration.Compositions comprising a agent of the invention formulated in acompatible pharmaceutical carrier may also be prepared, placed in anappropriate container, and labeled for treatment of an indicatedcondition. Suitable conditions indicated on the label may includetreatment of disorders or diseases, such as breast and ovarian cancersor other cancers and conditions associated with altered expression ofGT198.

B. Methods of Treatment

One embodiment provides a method for treating one or more symptoms ofcancer by administering an effective amount of an inhibitory nucleicacid specific for a nucleic acid encoding a GT198 variant protein toalleviate one or more symptoms associated with a cancer. The inhibitorynucleic acid can be antisense DNA or siRNA. Symptoms associated withcancer include tumor size and cellular proliferation. Representativecancers that can be treated include, but are not limited to ovarian,prostate and breast cancers. The inhibitory nucleic acid can be one ormore of the compositions disclosed above.

The disclosed GT198 or GT198 variant antagonist compositions can beadministered to a subject in need thereof alone or in combination withone or more additional therapeutic agents or combinations of the atleast two different GT198 antagonists. Representative GT198 or GT198variant antagonists include, but are not limited to inhibitory nucleicacids such as siRNA and antisense DNA, and antagonistic antibodies orantigen binding fragments thereof. The additional therapeutic agents areselected based on the condition, disorder or disease to be treated. Forexample, GT198 antagonists can be co-administered with one or moreadditional agents that function to inhibit GT198 biological function orbioavailability.

The GT198 antagonist can also be combined with one or more additionaltherapeutic agents. Representative therapeutic agents include, but arenot limited to chemotherapeutic agents and pro-apoptotic agents.Representative chemotherapeutic agents include, but are not limited toamsacrine, bleomycin, busulfan, capecitabine, carboplatin, carmustine,chlorambucil, cisplatin, cladribine, clofarabine, crisantaspase,cyclophosphamide, cytarabine, dacarbazine, dactinomycin, daunorubicin,docetaxel, doxorubicin, epirubicin, etoposide, fludarabine,fluorouracil, gemcitabine, hydroxycarbamide, idarubicin, ifosfamide,irinotecan, leucovorin, liposomal doxorubicin, liposomal daunorubicin,lomustine, melphalan, mercaptopurine, mesna, methotrexate, mitomycin,mitoxantrone, oxaliplatin, paclitaxel, pemetrexed, pentostatin,procarbazine, raltitrexed, satraplatin, streptozocin, tegafur-uracil,temozolomide, teniposide, thiotepa, tioguanine, topotecan, treosulfan,vinblastine, vincristine, vindesine, vinorelbine, or a combinationthereof. Representative pro-apoptotic agents include, but are notlimited to fludarabinetaurosporine, cycloheximide, actinomycin D,lactosylceramide, 15d-PGJ(2) and combinations thereof.

IV. Kits

Kits having a container housing a probe that specifically binds to GT198or GT198 variant proteins or nucleic acids is also provided. The kit canbe used to detect the presence of mutation in GT198 indicative ofcancer. In one embodiment the probe is antibody specific for GT198 orGT198 variant proteins. The antibody can be monoclonal, polyclonal,single strand, humanized, and chimeric or an antigen binding fragmentthereof. In another embodiment the probe is a nucleic acid probe,preferably at least 10 nucleotides in length that specifically binds toa nucleic acid encoding GT198 or a variant thereof. The kit optionallyincludes probes for detecting one or more biomarkers for cancer.Preferred biomarkers in cancer that can be combined with GT198 markerinclude mutations in BRCA1 and BRCA2. Thus, nucleic acid probes thatspecifically hybridize to mutations in BRCA1 and BRCA2 can be includedin the kit. Reagents such as buffers and materials to detecthybridization can also be included in the kit.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of skill in the artto which the disclosed invention belongs. Publications cited herein andthe materials for which they are cited are specifically incorporated byreference.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

EXAMPLES

Methods and Materials

Cloning of GT198 Variants.

The presence of human GT198 variants in tissues was initially detectedby RT-PCR, followed by sequencing analysis. Full-length GT198-1 to −4cDNAs containing a longer 5′ end similar to that of wild type weresubsequently obtained by 5′RACE (Invitrogen). Human and mouse GT198avariant cDNAs were detected from human BG01 cells and mouse P19 stemcells, respectively, both with the retention of the intron 1. Subsequent5′RACE using intron 1 primer showed a shorter 5′ end at the samenucleotide positions in human and mouse sequences almost immediatelybefore their start codons. Human GT198a-4 was identified in tissuesusing intron 1 specific primers. Expression pattern analysis of P19 stemcells then relied on the specific primer at 5′ for the wild type GT198and the intron 1 primer for variant GT198a. Identified cDNA sequenceshave been deposited and released in GenBank with accession numbers asfollows: hGT198, FJ952179; hGT198-1, FJ952180; hGT198-2, FJ952181;hGT198-3, FJ952182; hGT198-4, FJ952183; hGT198a, GQ851964; hGT198a-4,GQ851965; mGT198, FJ937966; and mGT198a, FJ937967.

RT-PCR and Quantitative PCR Analysis.

Endogenous GT198 and its variants were analyzed using first-strand cDNAsfrom multiple normal human tissues and cancer cell lines (MTC™ panels,Clontech). In stem cells, total RNA was isolated at each differentiationstage using Trizol reagent (Invitrogen), treated with DNase I,reverse-transcribed to cDNA using SuperScript III (Invitrogen), andnormalized for their concentrations before RT-PCR. Real-time PCR(iCycler, BioRad) was performed using SYBR Green dye in duplicate in a25 μl reaction. The results were normalized to GAPDH.

Stem Cell Differentiation.

Embryoid body formation from mouse embryonic stem (ES) cells orembryonal carcinoma (EC) P19 cells has been previously described Brooks,Y. S. et al. J Biol Chem, 284:18033-46 (2009). Briefly, undifferentiatedP19 cells (EC) were induced to differentiate by 500 nM all-transretinoic acid for 4 days to form embryoid bodies (EB2-4). Total RNA wasisolated at each stage of P19 differentiation for RT-PCR analysis. MouseES cells were grown on γ-irradiated feeder fibroblasts, and weredifferentiated into embryoid bodies by serum deprivation. ES-derivedembryoid bodies were paraffin-embedded for immunohistochemistryanalysis.

Luciferase Assay.

P19 cells were maintained in α-MEM supplemented with 2.5% fetal bovineand 7.5% bovine calf serum. Cells were cultured in 24-well plates andtransfected in triplicates using Lipofectamine 2000 (Invitrogen) withMMTV luciferase reporter (100 ng), glucocorticoid receptor (10 ng), andvarious amounts of GT198 expression plasmids. Cells were incubated withthe ligand dexamethasone (100 nM) to induce the MMTV-luciferasereporter, when applicable, for 16 hours before harvest. Relativeluciferase activities in were measured by a Dynex luminometer. Data areshown as means of triplicate transfections±standard errors.

Immunohistochemistry and Immunofluorescence.

Polyclonal anti-GT198 antibody was previously prepared from rabbits(Covance) (Ko, L., et al., Mol Cell Biol, 22:357-69 (2002). RecombinantGT198 or its fragments as antigens were cross-linked to the Affi-gel 10resin (Bio-Rad) for affinity purification. Paraffin-embedded tumortissues in array format were from Imgenex and US Biomax, Inc. Antibodybinding was detected using biotinylated anti-rabbit IgG F(ab)₂ secondaryantibody followed by detecting reagents (DAKO). Sections werecounterstained with hematoxylin. Immunofluorescence double staining wascarried out using rabbit anti-GT198 antibody and mouse anti-Flagantibody (Sigma). Cy3- and FITC-conjugated secondary antibodies (JacksonImmuno-Research Laboratory Inc.) were applied at a dilution of 1:200.GFP-Rad51 plasmid contains mouse full-length Rad51 in pEGFP-C3 vector(Clontech). For TUNEL assay, immunofluorescent antibody binding wasperformed prior to the TUNEL assay using an In Situ Cell Death DetectionKit (Roche). Sections were counterstained with DAPI.

Western Blot Analysis.

Endogenous or overexpressed GT198 and its variants were detected byWestern blot analysis using whole cell lysate from overexpressed 293cells or from P19 stem cells. Immunoprecipitation was performed usinganti-Flag M2 agarose beads (Sigma), incubated with 1:10 diluted cellextracts from transfected cells in binding buffer. The precipitates werewashed and subjected to Western blot analysis using anti-GT198antibodies. The blots were probed with anti-GT198 at a dilution of 1:200and detected with the ECL system (Amersham Pharmacia).

Whole Mount In Situ Hybridization.

Mouse embryos at E8.5, E9.5, and E10.5 were fixed overnight in 4%paraformaldehyde in PBS with 0.1% Tween at 4° C. and dehydrated througha serial methanol at 25%, 50%, 75% and 100%. The dehydrated embryos weretreated with RNase-free DNase I (50 U/ml) before hybridization withdenatured riboprobe. The antisense and sense riboprobes were produced byin vitro transcription in the presence of Digoxigenin-UTP (RocheDiagnostics), using full-length GT198 cDNA in pcDNA3 vector. Stainedembryos were fixed and photographed.

Subjects and Materials.

Genomic DNA or RNA was isolated from the Epstein-Barr virus(EBV)-immortalized B-lymphoblastoid cell lines derived from familialbreast cancer patients. These patients were diagnosed with breastcancers before age 40 and did not carry a mutation in BRCA1 and BRCA2.Genomic DNA isolated from whole blood was also available to confirm themutations identified in cells lines. Informed consent from theindividuals was obtained following institutional guidelines.

Southern Blot Analysis.

Genomic DNA (10 ug) isolated from B-lymphoblastoid cell lines of breastcancer patients was digested by restriction enzyme Pst I overnight,separated by 1% agarose gel, transferred to positively charged nylonmembrane, and probed with random-primed ³²P-labeled probe (Stratagene).The blot probed by exon 1-3 probe was stripped and re-probed with exon4-5 probe. Both probes contain small introns as illustrated in FIG. 4 a.

Example 1: Protein Sequence Homology Among GT198 and the BRC Repeats inBRCA2

GT198 has an N-terminal domain and a C-terminal DNA-binding domain (DBD)linked by a leucine zipper dimerization domain. Due to the functionalsimilarities among GT198, BRCA1, and BRCA2 described below, we performedthe multiple sequence alignment of BRCA1, BRCA2, and GT198 usingClustalW, and identified that the C-terminal DBD of GT198 is homologousto the BRC repeats in BRCA2 (FIG. 1). In particular, the C-terminalregion of GT198 is aligned with the third and the fourth BRC repeats ofBRCA2. The demonstrated Rad51-binding activity among these threesequences provides functional support for the presence of a potentialBRC repeat in GT198. The N-terminus of GT198 showed limited homology tothe BRC repeats. The functional implication of their sequence homologyis that GT198 could compete with BRCA2 in binding to Rad51 through theBRC repeat region as illustrated in FIG. 1 c.

Example 2: GT198 is Co-Expressed with BRCA1 and BRCA2

The endogenous nuclear expression pattern of GT198 is remarkably similarto or almost indistinguishable from both BRCA1 and BRCA2 expression inmice (Chodosh, L. A., J Mammary Gland Biol Neoplasia, 3:389-402 (1998)).GT198 protein expression increases in primitive ectoderm at four days ofembryoid bodies derived from embryonic stem cells. Consistent with theprevious Northern blot analysis (Ko, L., et al., Mol Cell Biol,22:357-69 (2002)), in situ hybridization revealed a marked increase inGT198 mRNA expression in neural tubes from E8.5 to E10.5 (data notshown). The expression peaks at E12.5 in all three germ layers and isdownregulated at E18.5. In adult rodents, GT198 protein waspredominantly found in testis with restricted expression in thymus,spleen and ovary by Western blot analysis (Ko, L., et al., Mol CellBiol, 22:357-69 (2002)). Similar to BRCA1, GT198 mRNA expression canalso be detected in different tissues. At the cellular level, GT198protein expresses in spermatocytes of the testis and oocytes of theovary, consistent with its essential function in testis and ovary sinceboth male and female GT198 knockout mice are sterile (Petukhova, G. V.,et al., Dev Cell, 5:927-36 (2003)). In ovary, GT198 has high levels ofnuclear expression in granulosa cells of the secondary and Graafianfollicles but not in the primordial follicles or the corpus luteum.Theca cells have low levels of nuclear expression. This pattern ispotentially associated with cycled steroid hormone action given thatGT198 interacts with steroid receptors and stimulates receptor-mediatedgene regulation (Ko, L., et al., Mol Cell Biol, 22:357-69 (2002)). Theremarkably similar patterns among GT198, BRCA1 and BRCA2 expression(Lane, T. F., et al., Genes Dev, 9:2712-22 (1995); Sharan, S. K. &Bradley, A., Genomics, 40:234-41 (1997); Durocher, F., et al., JHistochem Cytochem, 45:1173-88 (1997); Blackshear, P. E., et al.,Oncogene, 16:61-8 (1998)), suggest that they may have a close functionalrelationship in same developmental pathways.

Example 3: Alternatively Spliced GT198 Variants Inhibit Wild Type GT198

During RT-PCR analysis of human GT198 mRNA expression, a number of GT198alternative splice variants were identified. Alternative splicing ofGT198 is tissue-specific with more variety of splicing variants werefound in embryonic tissues or in cancer cells than in normal adulttissues. Sequencing results showed that all alternative splicing eventsoccur strictly at the GT-AG consensus sites, some of which are supportedby NCBI EST evidence. Interestingly, variations of alternative splicingalways occurred at the 5′ half of the gene, and all generate prematurestop codons with early termination of translation (FIG. 2a ). Allvariant transcripts contain an open reading frame encoding the DBD (aa126-217) with BRC repeat homology. A few variants also encode the Nterminus. These truncated forms of potential proteins are devoid of theleucine zipper at amino acids 89-117 (Ko, L., et al., Mol Cell Biol,22:357-69 (2002)), and thus would not form a dimer similar to the wildtype. In addition, two alternative transcriptional start sites differingby the 5′ at the first exon have been identified by 5′RACE. The longertranscripts at 5′ end encode the wild type as well as the variantsdesignated as GT198 1-4. The shorter transcripts at 5′ end always retainthe first intron and encode variants designated as GT198a and GT198a-4.The dual transcriptional start sites, present in both human and mouse,suggest the differential usage of promoters by wild type and variants.Of note, human BRCA1 is also known to have dual transcriptional startsites (hodosh, L. A., J Mammary Gland Biol Neoplasia, 3:389-402 (1998))although their linkage to each variant was uncharacterized due to itslarge gene size.

The functional importance of GT198 alternative splicing in a stem celldifferentiation system was then investigated. Variant GT198a with theretention of intron 1 was identified as a predominant variant form instem cells of both human and mouse. During retinoic acid-induced mouseP19 stem cell differentiation, a marked decrease in GT198a and anincrease in wild type GT198 mRNA were detected by RT-PCR and verified byquantification (FIGS. 2d-e ). Interestingly, we found that BRCA1 and itsvariant BRCA1Δ11b had a similar switched expression pattern as to GT198and GT198a (FIGS. 2b-c ), which is consistent with a previously reportedNorthern blot analysis of BRCA1 in mouse embryos (Hakem, R. et al.,Cell, 85:1009-23 (1996)). The data imply that dual promoters in bothgenes are differentially regulated during embryoid body formation.GT198a mRNA is more abundant than GT198 mRNA inferred by using commonprimers that detect both. At the protein level, however, neither GT198anor GT198-4 variants produces sufficient amount of variant proteins whendetected by Western blot using GT198 protein fragments as controls. Evenwhen abundant variant mRNA was present, only a trace amount of variantprotein corresponding to GT198 DBD with BRC repeat homology can bedetected only by immunoprecipitation when overexpressed in 293 cells orby immunofluorescent staining possibly due to the apoptosis oftransfected cells. Therefore, the nuclear protein detected byimmunohistochemical staining in normal tissues should be mainly wildtype GT198 protein. Surprisingly, when transcriptional activity wastested in P19 stem cells using a mouse mammary tumor virus (MMTV)promoter-luciferase reporter system, variants GT198a and GT198-4 showedrobust stimulation of transcription, while wild type GT198 repressed thetranscriptional activity of MMTV promoter (FIG. 3a-b ). The DBD with BRCrepeat homology and a leucine zipper deletion mutant that preventedGT198 dimerization also showed transcriptional activation. The Nterminus did to have significant activity. Consistent with the reportthat the DBD is required for GT198 activity (Enomoto, R. et al., J BiolChem, 279:35263-72 (2004)), the data suggest that the GT198 DBD with BRCrepeat homology, which was encoded by all variant mRNA, may compete withwild type GT198 and counteract its activity. Although detailedmechanisms need to be further elucidated, it is possible that the GT198splice variants may serve as natural dominant negatives. In addition tothe competition at protein level, the variants could prevent wild typemRNA expression through competitive alternative splicing or throughyet-to-be-identified downstream RNA interference. Importantly, similarregulation may exist for BRCA1 or BRCA2 if their inhibitory variants canbe thoroughly studied. Nonetheless, through potential multiplemechanisms, alternatively spliced variants of GT198 inhibit the wildtype.

Example 4: Overexpression of GT198 Splice Variants Block Rad51 FociFormation, Induce Apoptosis and Promote Cytoplasmic Translocation ofWild Type GT198

GT198 variants were tested for their effect on Rad51 foci formation uponionizing radiation. HeLa cells were cotransfected with GFP-tagged Rad51and with Flag-tagged constructs encoding GT198, variants GT198a,GT198-4, the N-terminal domain, the DBD with BRC repeat homology, or theleucine zipper deletion mutant. Cells were γ-irradiated to induce Rad51foci before analyzed by immunofluorescent staining. As compared to theuntransfected control, neither GT198 nor the N-terminus disrupted theRad51 foci, while all DBD-encoding proteins with BRC repeat homology,including the two GT198 variants, blocked the foci formation. The focidisruption caused a homogenous expression of Rad51 not only in thenucleus, but also diffusing into the cytoplasm. Interestingly, whilewild type GT198 is exclusively nuclear, all its fragments, mutants, orvariants had cytoplasmic expression. Dimerization may be important fortheir subcellular localization and that the GT198 dimer is involved inRad51-associated function (Pezza, R. J., et al., Genes Dev, 21:1758-66(2007)). Detailed examination of a large number of transfected cellsshowed that cells with disrupted Rad51 foci also have altered morphologywith shrinking nuclei. TUNEL assays confirmed that the DBD-containingmonomeric proteins including splice variants induced marked apoptosiswith DNA fragmentation, implicating that wild type GT198 is essentialfor the cell survival in culture. The N terminus showed cytoplasmicexpression without induction of apoptosis or disruption of Rad51 foci,possibly because the essential functional unit of GT198 requires its DBDwith BRC repeat homology. These data suggest that the BRC repeat regionthat competes or blocks GT198 normal activity. The loss of the wild typefunction triggers apoptosis. This conclusion is consistent with thetranscriptional studies described above and also with the reportedDNA-binding activity in which the DBD of GT198 is required (Enomoto, R.et al., J Biol Chem, 279:35263-72 (2004)). Variant overexpression wasexamined to determine if it influenced endogenous wild type GT198expression by using non-overlapping antibodies against either the N orthe C terminus. The results revealed a punctuated pattern of endogenousGT198 that changes levels through the cell cycle. BRCA1 expression isalso known to be regulated during cell cycle. When an antibody againstthe N terminus of GT198 is analyzed with the C-terminal tagged variants,endogenous GT198 became cytoplasmic under the induction of variantactivity. Cytoplasmic expression was also observed using an antibodyagainst the C terminus of GT198. In summary, overexpression of GT198variants promotes wild type GT198 cytoplasmic translocation, impairsRad51 foci formation and induces apoptosis.

Example 5: Overexpression of GT198 Variants in Human Breast and OvarianCancers and Mouse Tumor Models

BRCA1 is a tumor suppressor, and its functional loss predisposes tobreast and ovarian cancers. Cytoplasmic BRCA1 overexpression in breastcancers has been previously reported in a high percentage of cases(Al-Mulla, F., et al., J Histochem Cytochem, 53:621-9 (2005)),indicating the connection between increased variants and wild-typedeficiency. Indeed, many identified mutations of BRCA1 alter itssplicing. This prompted immunohistochemistry analyses of GT198expression in primary breast and ovarian cancers. Using the antibodyagainst full-length GT198, 238 cases of ovarian cancer and 114 cases ofbreast cancers were screened with normal controls in tissue arrays. Inovarian cancer cases, cytoplasmic expression of GT198 was found in 13.4%of stromal cells and in 29.8% of epithelial cells (Table 1). Normalhuman adult ovaries are devoid of cytoplasmic GT198 staining suggestingthe cytoplasmic expression is associated with cancers. Most of theovarian cancer cases except benign fibromas and fibrothecomas did notshow nuclear staining of GT198 (not shown). A characteristic high levelof cytoplasmic expression in exclusive stromal cells was found in mostof epithelial subtypes of ovarian cancers, including serous, mucinous,endometrial, clear cell carcinomas, and also in granulosa-theca cellcarcinoma. GT198 variant mRNA can also be detected in ovarian cancersand the cytoplasmic protein can be recognized by antibodies against boththe N- and C-terminus of GT198, suggesting that either variantexpression or wild type cytoplasmic translocation are present in tumors.In one case at early stage, the GT198 positive cells are located intheca cell areas surrounding the follicles while the follicles continuedto elongate and disintegrate into cord structures. When a large numberof cases were examined, it appears that at later stages of cancerdevelopment, accumulated granulosa cells underwent an epithelialtransition to evolve into epithelial types. GT198 positive cells aremost frequently found underlining the epithelium and at later stagesthey squeeze into channels of stromal areas when tumor mass becomessignificant. The GT198 positive cells are absent in metastatic tumorsthat originate from other sites suggesting the involvement of GT198 inovarian cancer development (Table 1). It is unknown whether these GT198positive stromal cells were related to theca cells. However, theirdistribution patterns in developing cancer tissues imply that thesepositive cells from each epithelial subtype might have the same origin.Supporting to the findings, considerable evidence has previouslydemonstrated that ovarian surface epithelium also known as germinalepithelium shares the same origin as follicles, and that oogenesisoccurs in ovarian surface epithelium (Nishida, T. & Nishida, N., ReprodBiol Endocrinol, 4:42 (2006); Bukovsky, A., et al., Endocrine, 26:301-16(2005)). The observation using GT198 as a marker is consistent with theevidence that hormone-responsive granulosa and theca cells may evolveinto epithelial types of ovarian cancers.

In the analysis of 114 cases of human breast cancers, cytoplasmic GT198expression was found in 17.5% of stromal cells and in 48.2% ofepithelial or myoepithelial cells (Table 1). Normal breast tissue isnegative for GT198 expression with the exception of a few positivestroma cells. Myoepithelial expression of cytoplasmic GT198 isfrequently found with hyperplasia or with disrupted ductal structure.Since GT198 regulates hormone-stimulated nuclear receptor signaling, itsalteration in myoepithelial cells supports the existing evidence thatthe hormone-responsive myoepithelial layer (Strum, J. M., Cell TissueRes, 193:155-61 (1978)), plays a critical role in breast cancerdevelopment (Gudjonsson, T., et al., J Mammary Gland Biol Neoplasia,10:261-72 (2005)). Furthermore, in mouse tumor models carrying MMTV-Rasor the polyomavirus middle T antigen transgenes, GT198 has massivecytoplasmic expression in the stroma of mammary gland tumors, ascompared to the nontransgenic normal tissue, even before morphologicalhyperplasia occurs. This data indicates that viral oncogene activationinduces GT198 variant overexpression. Since GT198 variants stimulate theMMTV promoter (FIG. 3a-b ), positive feedback between oncogene Ras andGT198 variant expression may facilitate tumor initiation. A cytoplasmicto nuclear GT198 transition occurs only at later stages when transformedtumor cells accumulate.

In summary, breast and ovarian cancers contain GT198 positive cells withcytoplasmic expression. The increased GT198 variant activity potentiallycauses wild type functional deficiency and disrupts differentiation ofcells that are survived from apoptosis. To further confirm this notion,nude mouse mammary glands were injected with mouse P19 stem cells stablytransfected with either wild type GT198 or variant GT198a. The datasuggested that variant GT198a but not wild type GT198 transfected cellsinduced significant tumor growth. The collective evidence in humanovarian and breast cancers and mouse tumor models suggests that GT198variant overexpression is an early event during tumor development, whichreflects the functional connection between GT198 and BRCA1.

TABLE 1 Cytoplasmic Expression of GT198 in Human Breast and OvarianCancers Stromal Epithelial Total n expression n (%) expression n (%)Ovarian cancer subtype Serous 112   7 (6.3)   45 (40.2) Mucinous 33   16(48.5)   14 (42.4) Endometrioid 11   3 (27.3)   6 (54.5) Clear cell 15  1 (6.7)   4 (26.7) Brenner 1 0 (0) 0 (0) Undifferentiated 7 0 (0) 0(0) Granulosa-theca 12   5 (41.7)   1 (8.3) Sertoli-Ledig 1 0 (0) 0 (0)Fibroma-thecoma 14 0 (0) 0 (0) Dysgerminoma 10 0 (0) 0 (0) Embryonal 3 0(0)   1 (33.3) Muellerian 3 0 (0) 0 (0) Metastasis from distance 16 0(0) 0 (0) Normal 14 0 (0) 0 (0) Total case of tumor 238   32 (13.4%)  71 (29.8%) Breast cancer subtype Invasive ductal 64   12 (18.7)   34(53.1) Invasive lobular 11 0 (0)   4 (36.4) Medullary 4 0 (0)   1 (25.0)Carcinoma in situ 6   2 (33.3)   4 (66.7) Hyperplasia 9   1 (11.1)   7(77.8) Fibroadenoma 3 0 (0) 0 (0) Lymph node metastasis 17   5 (29.4)  5 (29.4) Normal 4 0 (0) 0 (0) Total case of tumor 114   20 (17.5%)  55 (48.2%)

Example 6: Germline Mutations of GT198 in Early Onset Breast CancerPatients

To seek genetic evidence for the involvement of GT198 in breast cancer,GT198 mutations in genomic DNA and RNA isolated from immortalized bloodlymphocytes from nine early onset (<40 years) breast cancer cases whomwere negative for BRCA1 and BRCA2 mutations. Southern blot, real-timePCR, genomic walking, RT-PCR, and sequence analyses were performed.Southern blot analysis showed the presence of abnormal bands in case 3and case 9. Subsequent sequencing confirmed the allelic point mutationsat restriction sites in these two cases. In case 3, the C to G mutationis at 31 bp after the stop codon in 3′ UTR, c.*31C>G. In case 9, a C toT mutation in intron 4, 88 bp away from the splicing junction wasidentified, c.337+88C>T. Excluding the large intron 3, all exons andintrons were sequenced in these nine cases. A mono-allelic C to Tmutation (Q104Stop, c.310C>T) was identified in exon 4 of case 1. Thisnonsense mutation generates a premature stop codon and is predicted toprevent the translation of full length GT198 but permit the translationof the variant protein containing the BRC repeat homology encoded byexons 5-8. The impact of these sequence alterations on alternativesplicing was evaluated by RT-PCR. The results indeed suggested theincreased variant mRNA expression in immortalized lymphocytes in caseswith mutations, although detailed mechanisms remain to be studied.GT198a variant is markedly increased in case 1 and case 3, and GT198-4variant is increased in case 9. The variant cDNA of case 1 and case 3were found to carry the same mutations. Real-time PCR analysis did notdetect copy gain or loss of GT198 in these nine cases (not shown). Inaddition, two SNPs were found in intron 4 during the sequencing analysis(FIG. 4b ). Based on the HapMap database at NCBI, these two SNPs arecommon SNPs and are unlikely to be strong susceptibility alleles. TheGT198 gene covers two haploblocks joined at its large intron 3containing multiple Alu and L2 repeats (FIG. 4a ), however, we have notdetected any rearrangement in these nine cases by real-time PCR andgenomic walking analyses. More unidentified mutations could exist toaffect alternative splicing since the promoter/enhancer regions have notbeen analyzed. The family history of cases carrying mutations wasfurther examined and it was found that multiple individuals in eachfamily have cancers including breast cancer in a first degree relative(FIG. 4c ). The case 1 sister had breast cancer at the age of 40 and DNAwas available at the time of this report. Sequence analysis showed thatshe also carried the same c.310C>T (Q104Stop) mutation. These datacollectively indicate the presence of germline mutations of GT198 thatpromote GT198 variant expression in early onset breast cancers.

I claim:
 1. A method comprising detecting the presence or absence ofcytoplasmic mutant GT198 protein consisting of SEQ ID NO:13 in a humancancer patient's biological sample.
 2. The method of claim 1, comprisingdetecting GT198 gene mutations, copy number changes, or rearrangementsthrough performing a direct DNA sequencing, fluorescent in situhybridization, gel electrophoresis or polymerase chain reaction-basedanalysis.
 3. The method of claim 1, wherein cytoplasmic mutant GT198 isdetected with an anti-GT198 antibody by immunohistochemistry.
 4. Themethod of claim 1, wherein the patient's biological sample is selectedfrom the group consisting of blood, tissue, and cells.
 5. A method ofdiagnosing and treating breast cancer and gynecological cancercomprising: detecting the presence or absence of cytoplasmic mutantGT198 protein consisting of SEQ ID NO:13 in a human cancer patient'sbiological sample; diagnosing the patient with breast cancer andgynecological cancer when the cytoplasmic mutant GT198 proteinconsisting of SEQ ID NO:13 is present, and administering an effectiveamount of anti-GT198 antibody to the diagnosed patient, wherein theanti-GT198 antibody is selected from the group consisting of monoclonal,polyclonal, single strand, humanized, and chimeric antibodies.